Method for fluorescence analysis of white blood cells

ABSTRACT

A dye composition for differential analysis of white blood cells comprising a hypotonic aqueous solution of a metachromatic fluorochrome dye, e.g., acridine orange. The solution has a pH factor within normal physiological ranges for human blood. The white cell analysis is made by straining the cells with the fluorescent dye by suspending a sample of fresh blood in the dye solution, subjecting the suspension before dye uptake equilibrium is reached to radiation from a light source (e.g., radiation from a blue laser) having a wave length within the range of absorption of the dye, distinguishing the white cells from other blood particles by detecting fluorescenses (e.g., green fluorescences) emitted from the white cells in response to the radiation, and further distinguishing the various types of white cells by measuring the magnitudes of their respective fluorescence emissions (e.g., red and green fluorescences). Differences in the rates of uptake of fluorochrome dye of the various kinds of white cells are accentuated by altering the aqueous dye solution so that it becomes hypotonic.

United States Patent [191 Adams METHOD FOR FLUORESCENCE ANALYSIS OF WHITE BLOOD CELLS [75] Inventor: Lawrence R. Adams, Mahopac,

[73] Assignee: Bio/Physics Systems, Inc., Mahopac,

[22] Filed: Oct. 30, 1973 [21] Appl. No.: 410,994

Primary Examiner-Ronald L. Wibert Assistant Examiner-Matthew W. Koren Attorney, Agent, or Firm-Pennie & Edmonds [451 May 13, 1975 [57] ABSTRACT A dye composition for differential analysis of white blood cells comprising a hypotonic aqueous solution of a metachromatic fluorochrome dye, e.g., acridine orange. The solution has a pH factor within normal physiological ranges for human blood. The white cell analysis is made by straining the cells with the fluorescent dye by suspending a sample of fresh blood in the dye solution, subjecting the suspension before dye uptake equilibrium is reached to radiation from a light source (e.g., radiation from a blue laser) having a wave length within the range of absorption of the dye, distinguishing the white cells from other blood particles by detecting fluorescenses (e.g., green fluorescences) emitted from the white cells in response to the radiation, and further distinguishing the various types of white cells by measuring the magnitudes of their respective fluorescence emissions (e.g., red and green fluorescences). Differences in the rates of uptake of fluorochrome dye of the various kinds of white cells are accentuated by altering the aqueous dye solution so that it becomes hypotonic.

23 Claims, 2 Drawing Figures z :18 Y=Y :5 0 E33 DJ 23 Y=O qu.

AMP. 0F RED FLUORESCENCE PNEME mm 31% FIG. 1

TELETYPE ENCE X=X X AMF! OF RED FLUO METHOD FOR FLUORESCENCE ANALYSIS OF WHITE BLOOD CELLS fit. RUROUND OF THE INVENTION This invention relates to an improved composition for staining white blood cells, and to an improved method and apparatus for utilizing that composition in analyzing blood for white cells.

Blood is a fluid. circulating tissue found in all higher animal and in many invertebrates. it is a tissue, just as a skin, muscle and bone are tissues, because it contains living cells and has specific functions, chief among them being the conveyance of materials from one part of the body to another. The general principle on which the chemical life of the body is conducted is that each living cell carries out within itself all the chemical processes necessary to its existence. Therefore, all the materials which each cell requires must be carried to it, and those which it discards must be removed. Throughout the bodies of higher animals a highly specialized system of transport-the blood vascular system-has evolved, affording an efficient route for the blood and providing the necessary, intimate contact thereof with every living cell.

The principal materials which a living cell requires are sugar, amino acids, fats, vitamins, oxygen, salts, hormones and water. The organs of digestion convert the solid constituents of food into forms that the blood can absorb and deliver to the cells of the body. The principal substances which the cell must dispose of are carbonic acid and simple soluble compounds of nitrogen. These are conveyed by the blood to the various organs functioning in an excretory capacity, ridding the body of noxious wastes.

In all the higher animals blood consists of an aqueous fluid part, the plasma, in which are suspended corpuscles of various kinds: the red blood cells (erythrocytes), the white blood cells (leukocytes) and the blood platelets.

The plasma has a faint straw color and is clear unless a meal containing fat has been eaten recently, in which case it is somewhat milky because of the minute globules of fat which it transports. The two materials dissolved in greatest quantity are albuminous substances (proteins) and common salt.

The general nature of plasma resembles that of raw egg white, diluted with 0.9% solution of salt. In detail, its composition is roughly as follows: water, 90%; proteins (fibrinogen, globulins, albumin), 9%; salts (of Na, K, Ca, Mg, Fe, Cu, etc.), 0.9%; sugar, urea, uric acid and creatinin, traces. Water is present primarily in order to dissolve the other substances and to afford the blood a degree of fluidity sufficient to secure its easy flow through the minute capillaries of the vascular system.

Sodium chloride in blood plasma serves primarily to effect protein dissolution. Since most proteins in the blood do not dissolve in pure water, and since protein can form the basis of living material only if it is in solution, salt is essential.

The concentration of hydrogen ions in normal plasma equals 0.4 X 10" gram of hydrogen per liter (pH=T.4). Any considerable increase in hydrogen ions (lowering of pH value, or acidosis) causes increased and violent breathing; this in turn enhances the expul sion of carbonic acid from the plasma into the air, -wiing o raise the pH value of the blood to its normal level. On the other hand, should the blood become unduly alkaline (alkalosis) the kidney will relieve this condition by secreting an increased quantity of alkali in the urine. This balance of acid and alkali, the maintenance of which preserves the reaction of the blood at a pH of 7.4, is called the acid-base equilibrium of the blood.

The cells or corpuscles of the peripheral blood (i.e., blood outside the bone marrow) are divided into two groups: the red blood cells (erythrocytes) whose primary object is to transport oxygen and the white blood cells (leukocytes) whose functions include the engulfing and destruction of microorganisms and other foreign bodies in the blood stream (phagocytosis), In addition to these cellular elements, the blood contains socalled blood platelets (thrombocytes).

It is well known that mature white blood cells nor mally exist in the blood in different forms which can be classified into major groups. Each group forms a percentage of the total within the general limits for normal blood as shown in the following table:

Polymorphonuclear leukocytes Neutrophils: 38 to percent Eosinophils: l to 5 percent Basophils: 0 to 2 percent Mononuclear leukocytes Lymphocytes: 15 to 45 percent Monocytes: l to 8 percent The polymorphonuclear leukocytes not only have segmented nuclei, but are also characterized by having granules in the cytoplasm, and they are therefore sometimes referred to as granulocytes. Granulocytes develop in the bone marrow from cells called myeloblasts, which are undifferentiated and will mature into either neutrophils, eosinophils or basophils. The stages of growth of immature granulocytes have been -classified and the appearance of them characterized. Although immature granulocytes are present in low numbers in normal blood, their appearance in significant quantity is particularly significant for the diagnosis of infection or leukemia. A more detailed description of the various types of blood cells sufficient for a further understanding of the background of the present invention can be found in L. W. Diggs et al., The Morphology of Human Blood Cells, published in 1970 by Abbott Laboratories, and in M. M. Wintrobe, Clinical Hematology, 224-60 (6th ed. 1967) Even though they are far outnumbered by the red blood cells (erythrocytes) by a ratio of approximately 700 to l, the leukocytes are extremely important to the body in fighting disease and infections. Furthermore, probably because of that function, it has been observed that abnormal body conditions such as disease or infection often results in marked changes in the leukocytes in the blood stream. These changes can include a marked increase (leukocytosis) or decrease (leukopenia) in the total number of white cells in proportion to the number of red cells in the blood stream. Also, marked changes in the proportions of different types of leukocytes in the blood stream have been found to be characteristic and unique with respect to particular diseases. A detailed description of this phenomenon appears in M. M. Wintrobe, Clinical Hematology, 260-94 (6th ed. 1967). Thus, a total white cell count (ie, a count of the total number of white cells within a specified volume of blood) and a differential blood analysis (i.e., a count which reveals the relative percentages of the different types of white cells in the blood) are two extremely valuable diagnostic and medical research procedures.

The usual medical laboratory procedure for obtaining a quantitative or total white cell count is to combine a precise volume of a blood sample with a precise volume of a solution (lysing agent) which destroys the red cells, and then to place a portion of the sample within a counting chamber microscope slide and to visually count the number of white cells appearing in several squares of the counting chamber. The result is usually expressed in terms of the number of white cells per cubic millimeter. This is a tedious procedure and in volves making a count on such a small sample that inaccuracies are very likely to occur. Furthermore, the skill of the technician is extremely critical in achieving accurate results.

In the conventional medical laboratory procedure for obtaining a differential analysis of white cells within a blood sample, a smear of the blood sample is dried upon a clean glass microscope slide and is then treated with an appropriate reagent which typically contains a fixative such as methyl alcohol in combination with a mixture of stains. Alternatively, the preparation procedure may include treatment with the fixative as a separate step. The treated blood smear is then examined through a microscope under oil immersion. Sample counts are taken visually in various different areas of the blood smear and recorded. An even distribution of the white cells is difficult to obtain and therefore it is recommended that counts be taken at the edges of the blood smear as well as at the central portion, and that a minimum of 200 cells be counted. It is by this means that a differential analysis of white blood cells is normally made. Unfortunately, this method involves many difficulties and limitations which seriously affect the accuracy, including lack of uniformity in the blood smear sample, the extremely small size of the sample actually counted, the tediousness of the task (which en courages the laboratory technician to cut corners and to make short counts), and the inconsistencies in the skills of different technicians in recognizing and accurately recording all of the unique cells which he actually sees.

Because the aforementioned manual procedures are time consuming even for a highly skilled technician, the cost of each analysis is necessarily high, and the speed at which urgent tests can be obtained is limited.

As a result of the aforementioned problems associated with manual procedures for counting white blood cells and differentiating the various types thereof, machine or instrumental methods have been devised with a view toward accomplishing these tests automatically and on live blood.

In the case of machines intended for making quantitative white cell counts, these devices have generally involved the procedure of providing a blood sample suspension having a lysing agent which is adjusted so as to destroy the red cells without destroying the white cells. All of the remaining cells are then counted, and the assumption is made that they are all white cells. This prior machine method leaves much to be desired because the destruction of the red cells is not always complete, and any remaining red cells are then counted as White cells, thus hurting the accuracy of the count. Also, the condition which destroys the red cells may damage the white cells, and such damage often interferes with the subsequent identification of the white cells.

In the case of machines intended for making differential analyses of living (vital) white blood cells (see, for example, M. Ingram et a]. Scientific American, November, 1970, page 72-82), it has not been possible in the past to devise a satisfactory instrument except as described in US. Pat. No. 3,684,377 issued to the same assignee as the present invention; M. R. Melamed et al., European J. Cancer, 9, l81-l84 (Pergamon Press, 1973); M. R. Melamed et al., Am. J. Clinical Pathology, 57(1), -102 (January, l972); M. R. Melamed et al., Cancer, 29(5), l36l-68 (May, 1972); and L. R. Adams et al., Acta Cytologica, 15(3), 289-91 (1971). The aforementioned patent and articles, in particular, disclose for the first time the machine differentiation of vital white blood cells as between lymphocytes, monocytes, and the general class of polymorphonuclear leukocytes (granulocytes). However, it has hiterto beem impossible to instrumentally differentiate further among the various forms of granulocytes (i.e., basophils, eosinophils and neutrophils) as well as among mature and immature forms of white blood Cells and particularly to distinguish, when present, the immature forms of granulocytes. According to the above-cited Abbott Laboratories and Wintrobe references, the immature forms of granulocytes include as a general class the so-called myelocytes.

The ability to quantify living white blood cells and to differentiate them into six major categories (i.e., lymphocytes, moncytes, neutrophils, eosinophils, basophils and immature granulocytes (myelocytes)) is highly desirable for the purpose of diagnosing many bodily ailments and diseases which manifest themselves in ab normal proportions of the aforementioned six catego ries or leukocytes. Therefore, an automatic machine or instrumental technique for achieving the foregoing white blood cell count and differentiation rapidly and accurately would provide the physician with an important diagnostic and prognostic tool for management of the ill patient.

Accordingly, it is an object of the present invention to provide an improved automatic machine and method for accomplishing a differential analysis of white blood cells which is characterized by a high degree of accuracy and very low cost.

Another object of the invention is to provide an improved machine and method for producing a differential count of white blood cells with rapid speed of analysis (less than 1 minute as compared to 10 minutes or more by previously known procedures).

Another object of the invention is to provide an improved machine and method for producing a differential count of white blood cells under conditions in which the cells are shocked by exposure to a non physiologic medium during staining.

Another object of the invention is to provide an improved machine and method for producing a differential count of white blood cells which rely on the use of properties which are dependent on the rate at which the leukocytes develop elicitable fluorescence when exposed to a non-physiologic medium.

Another extremely important problem in devising a machine method for obtaining a white blood cell differential count is that of providing signals from the cells to the machine to enable it to recognize and distinguish white cells from all other bodies within the blood such as red cells or platelets. The machine recognition and discernment of all white cells is essential in the problem of classifying them so as to provide the desired differential analysis.

Accordingly, another object of the present invention is to provide an improved composition for staining white blood cells, and for generating a signal therefrom, and an improved machine method for recognizing thereby and distinguishing all white cells within 3 l0 blood sample to obtain a white cell differential analysis.

SUMMARY OF THE INVENTION The foregoing objects are achieved according to the 15 present invention by the discovery of an improved blood cell staining composition and an apparatus and procedure for employing same which provide a clear basis for rapidly distinguishing the aforementioned six different categories of vital white blood cells (including 20 immature granulocytes) in a machine analysis.

The staining composition of the present invention comprises an aqueous solution of a metachromatic fluorochrome dye (i.e., a dye which undergoes fluorescence at a multiplicity of wavelengths in response to excitation by radiation within its range of absorption) such as acridine orange. This solution is formulated so that the pH thereof is at the normal physiological level of approximately 7.4. However, the solution is made hypotonic, the osmolarity or salinity thereof being gen- 3 ment for a period of time lasting from about 10 seconds to several minutes following the addition of the staining composition to the blood sample, depend on the nature of the particular white cell undergoing fluorescence,

i.e., depending on whether the cell is a lymphocyte, 60

monocyte, basophil, neutrophil, eosinophil or immature granulocyte. Without wishing to be bound by theory, it is believed that this phenomemon is due to the hypotonic nature of the staining composition which unexpectedly causes the rates of dye uptake by the white cells to differ among the cellular organelles of the aforementioned six types. In other words, the different amplitudes of fluorescences among the various cell types are determined by the rate-controlled degree of dye uptake which differs from cell type to cell type.

Generally, the staining composition of the present invention is hypotonic to the extent that the concentration of NaCl therein is between about 2.5 and 7.5 grams per liter of solution. We have preferred to use 4.25 grams per liter of solution. Alternatively, other well-known equivalents of sodium chloride for this purpose, e.g., sodium citrate, can be used.

In the case where acridine orange is employed as the dye component of the staining composition, it is generally used at a concentration on the order of four times that disclosed in the aforementioned U.S. Pat. No. 3,684,377. In other words, the concentration of acridine orange in a freshly prepared staining composition is sufficient to provide a concentration of the dye in the blood sample suspension to be tested of between about 4 X l0 and about 4 X 10 grams per liter of suspension. The use of such unusually high dye concentrations has been found for some unexplained reason, to enhance the degree of rate dependence of dye uptake by the various types of white blood cells.

In a preferred mode of carrying out the method of the present invention, a fresh blood sample is suspended in the aforementioned hypotonic aqueous acridine orange solution and the resulting suspension is preferably al- 5 lowed to stand for a period of up to about 30 to lOO seconds, preferably with mild stirring during that time, The suspension is thereafter subjected to radiation from a blue laser (488 millimicron wavelength) and the cells are differentially classified on the basis of the differences in the magnitudes of red and green fluorescences emitted from individual cells in response to excitation from the blue laser radiation.

The dye acridine orange or euchrysine, which is an important constituent of the preferred staining composition of the present invention, is sometimes referred to in abbreviated form as simply A0. Its utility in the method of the present invention stems from the fact that it possesses the properties of a metachromatic flurochrome. This material is an organic compound for which the chemical name is 3,6-bis-(dimethylamino)acridiniumchloride and whose structural formula is:

NCCII II Acridine orange is also identified by color index specification 46,005 from the publication entitled COLOR INDEX, Second Edition-of 1956 and l957, published jointly by the Society of Dyers and Colorists of Great Britain, and by the American Association of Textile Chemists and Colorists of Lowell, Mass. Acridine orange is commercially available, e.g., from Eastman Kodak Company, Rochester, NY,

Acridine orange has been recognized for some time as a fluorescent stain which is capable of staining nucleic acids such as ribonucleic (RNA) and deoxyribonucleic acid (DNA). For instance, see Rudolf Riglers articles entitled Microfluorometric Characterization of Intracellular Nucleic Acids and Nucleoproteins by Acridine Orange" in Volume 67, Supplementum 267 of Acta Physiologica Scandanavica (Stockholm, 1966) and Acridine Orange in Nucleic Acid Analysis in Volume I57, Article 1 of Annals oft/1e New York Academy ofSci'ences of Mar. 31, 1969, pages 21 l224. The application of acridine orange in staining cells is disclosed in US. Pat. No. 3,497,690 to Wheeless et al. However, the novel method of the present invention in which this material is used in the unique way described herein as a supravital metachromatic fluorescent dye for white blood cells has never been recognized. Since it is regarded primarily as a red fluorescence dye for RNA, and since the amount of RNA is generally considered negligible in white blood cells, acridine orange has not been considered as a likely candidate for providing meaningful information concerning the aforementioned six classes of leukocytes However, it has cen discovered that the unexpected utility of acridine range in this regard is achievable according to the method of present invention and thereby permits the automatic machine discernment of white blood cells from all other blood particles and the differentiation of leukocytes into lymphocytes, monophils, basophils, neutrophils, eosinophils and myelocytes (immature granulocytes).

Although the method and apparatus disclosed herein are described with particular reference to using euchry sine or acridine orange, it is understood that other suitable fluorochromes (i.e., dye stains possessing the attributes of a metachromatic fluorochrome) may be used consistent with the scope of the present invention.

As mentioned above, the preferred staining composition comprises acridine orange in a hypotonic aqueous solution at a concentration of between 4 X l and 4 X grams of acridine orange per liter of solution. The solution includes other additives to provide an osmolarity below the normal physiological ranges for human blood plasma. In the preferred concentrations of acridine orange solution, the mixture may not be a true solution, but partly a suspension of aggregates of dye molecules or, perhaps more properly, a colloidal dispersion, in which extremely minute undissolved particules are suspended in the liquid. However, this composition is referred to as a solution throughout this specification. The mixture of the acridine orange solution with a blood sample is then referred to as a suspension. Thus, while the acridine orange solution may not be a true solution, the use of that term serves to distinguish the staining composition per se from the liquid suspension formed after the addition of the blood sample.

The normal physiological pH value for human blood is generally considered to be 7.40, plus or minus 0.05 pH unit, and the pH of the staining solution is likewise preferably adjusted, within practical limits, to 7.40, plus or minus 1 pH unit. On the other hand, the osmolarity, which is a function of the concentration of salts in the solution, is adjusted to below the physiological level of approximately 8.5 grams of sodium chloride per liter of solution, whereby the medium becomes traumatic to the blood cells.

In the description of Example 1, below, a procedure is followed in which separate solutions of acridine orange and the saline buffer combination are produced and then combined. The combined solution tends to form colloidal particles which apparently include the acridine orange dye. It has been observed that the combined solution containing the colloidal particles appears to have a rather limited shelflife. Tlr. colloids up pear to precipitate out upon the walls and bo.tom ct the container. Accordingly, it is preferred to store the solutions separately, one solution containing only acridine orange, and the other solution containing only the saline buffer combination. The two solutions are then combined in the proper proportions on the day when they are to be used.

Whether the two solutions are stored separately, or in a combined solution, it has been observed that there appears to be an aging process which ocurs such that higher acridine orange concentrations may be necessary to achieve the desired results with older solutions as compared to relatively fresh solutions.

Without wishing to be bound by theory, it is believed that the high degree of effectiveness of the composition and method of the present invention has to do with the fact that there is competition for the dye among the three compartments: solution, granules and nucleus. The rate of dye uptake may also be mediated by the cell membrane. The stain solution of the present invention promotes large differences between the various types ofleukocytes in the amounts of dye taken up by the cell nuclei and granules. Additional factors may involve shielding effects of the granules on the nucleus and differences in the chemistry of the granules and their numbers in each cell type.

Furthermore, it is believed that the reason for the distinctive fluorescence (green) which distinguishes the white cells from other blood particles such as erythrocytes is that the dye combines with the DNA (deoxyribonucleic acid) of the cell nuclei in a distinctive way such that a green fluorescence can be elicited by excitation with blue laser illumination. The major portion of the uptake of dye by each cell, which does not combine with the nuclear DNA, appears to stain the cytoplasm, and is particularly concentrated in the cytoplasmic organelles known as granules. It is believed that this is the reason why the granulocytes, the white cells which are particularly distinguished by the presence of granules in the cytoplasm, apparently take up more of the acridine orange dye in the cytoplasm, and thus provide a greater red fluorescence signal. All of the acridine orange dye taken up by the cytoplasm (as distinguished from that taken up by the nuclei) appears to cause red fluorescence in response to the blue laser light. It is a feature of the present invention that, although the green tluorescence is due to staining of DNA at equlibrium the cells are measured before equilibrium is reached and differences in green fluorescence result from the stain depletion by the granules or shielding of light by them. The upper and lower populations of granulocytes merge at longer staining times.

Further objects, advantages, and features of the invention will be apparent from the following description, claims and the accompanying drawings wherein:

FIG. 1 is a schematic diagram of an apparatus which can be employed in carrying out the machine method of the invention.

FIG, 2 is an illustration of a cluster display of six categories of white blood cells which can be produced according to the method of the present invention.

DESCRIPTION OF THE DRAWINGS A preferred apparatus for carrying out the method of the present invention, particularly with respect to the sample flow and optical systems is constructed in accordance with the teachings contained in a prior application Ser. No. 2,750 filed .lan. ll, l970 by Mitchell Friedman, Louis A. Kanicntslty, and Isaac Klinger for PHOTOANALYSlS APPARATUS, now U.S. Pat. No.

3,705,771, and assigned to the same assignee as the present application. Other features of a preferred apparain for carrying out the method of the present invention. and particularly relating to the arrangement of the counters, cathode ray oscilloscope apparatus and associated circuits, are carried out in accordance with the teachings of another prior patent application Ser. No. 25.93l filed Apr. 6, 1970, by Louis A. Kamentslty and lsauc iilirtger for a PARTICLE ANALYSIS METHOD AND APPARATUS, now U.S. Pat. No. 3,662,77l, and assigned to the same assignee as the present application as well as in the above-mentioned U.S. Pat. No. 3,684.377. The disclosures of both of these prior patent applications and patent are incorporated herein by reference. However, for the sake of completeness, portions of the disclosures of these references are reproduced here, and specifically related to the process of the present invention.

Referring particularly to P16. 1, there is illustrated a schematic diagram of an apparatus which may be employed in carrying out the method in accordance with the present invention. The apparatus includes an optical chamber 10 containing a microcuvette 11 through which a stream 12 of cells may be passed while entrained in the suspension and supplied through a sample tube 14 from a reservoir (not shown). The suspension is preferably surrounded by a sheath of water 15 to confine the particles (cells) to a very fine stream. As the stream 12 of particles passes through the chamber 10, it passes through a narrow beam of light from a light source 22. Light source 22 is preferably an argon laser, and may include a cylindrical lens 23 for shaping and directing the light.

Different optical reactions of the individual blood cells to the light beam 20, in the form of fluorescent radiation from each cell, are detected by photoelectric pick-up elements 24 and 26, which can be photomultiplier tubes. The signals detected by the photoresponsive pick-up elements 24 and 26 are converted by those elements to electrical signal pulses which are supplied through connections 30 and 32 to solid state amplifiers 34 and 36, respectively. The pick-up element 24 is arranged to respond to red fluorescence signals, and element 26 is arranged to respond to green fluorescence signals. The transmission of optical fluorescence signals to the pick-up elements is enhanced by condensing lens 16. A dichroic mirror 18 is provided which has a nominal cut-off wavelength for light at about 5,800 angstrom units. Thus, it reflects light of all wavelengths below that limit, through a filter 18A, to the pick-up 26. All optical signals above 5,800 angstroms in wavelength are transmitted through the dichroic mirror 18, and through an optical filter 188 to the pick-up 24. The pick-up 24 receives the red fluorescence signals and the filter 183 passes a red band of radiation in the neighborhood of 6,300 angstrom units wavelength. Similarly, the pick-up 26 receives the green fluorescence signals and filter 18A passes a green band of radiation in the neighborhood of 5,300 angstrom units wave length.

The red and green photomultiplier signals, after amplification in amplifiers 34 and 36, respectively, are sampled and held in sample-hold amplifier-circuits'38 and 40. respectively, to produce two electrical pulses which are proportioned in amplitude to the magnitudes of the two fluorescent amplitudes. A threshold circuit 42 is c nnec ed to the green photomultiplier signal at 4.. and roduces a control signal pul e when the green fluo escenc is above a t'n'crl \mluc wliczttivc Li leukocyte, and activates the sample-hold amplifier circuits 38 and 40 and timing signals for the analog-digital converters 44 and 45 and computer 46. The two-held fluorescence pulses are each converted to afdigital representation of thefluo'rescence'values'by ihe two analogdigit'al-cohverters 44 and 4S producing two eight-bit members in two digital registers 48 and 50. Digital registers 48 and 50 are connected in turn to the input data buss of computer 46. An oscilloscope 52 monitors the outputs of the sample-hold circuits 38 and 40 to produce a display on the screen 54 of the oscilloscope. A second oscilloscope (not shown) can also be used which is controlled by the computer 46 to monitor computer-processed displays.

The computer 46 processes the signals from analogdigital converters 44 and 45 as well as input from the operator via a teletype 56 and produces an output indicating the differential count.

FIG. 2 depicts a cathode ray tube display of a white cell analysis characteristically produced in accordance with the methods of the present invention, and with particular reference to Examples Xi and Xll. ln this display, the green fluorescence signals have been used for vertical deflection up from the bottom margin (origin), and the red fluorescence signals have been used for horizontal deflection to the right from the origin line on the left margin. Typical clusters of bright spot signal points for groups of cells are indicated at 60, 70, 80, 90, and which correspond respectively to lymphocytes (L), monocytes (M), neutrophils (N), basophils (B), eosinophils (E) and immature granulocytes US).

In order to avoid any possibility of the detection of false signals, the signal circuit for the detection of the green fluorescence is preferably operated with a threshold circuit, as indicated for instance by the horizontal threshold line Y so that only signals having a sufficient green fluorescence signal value to exceed the threshold line Y, are actually registered and indicated in the visual display. Similarly, an upper threshold limit may be established, as indicated at Y2. By the employment of additional threshold limits at positions as indicated by lines X X1, X2, X3, X4 and X5 individual point clusters may be picked out and displayed alone, or counted as mentioned below in Example Xll, For instance, cluster 70 may be selected by setting the upper limits at Y2 and X3 and the lower limits at Y1 and X1.

in using the apparatus shown schematically in FIG. 1 to obtain the cathode ray tube display of FIG. 2, the computer 46 first monitors the values of green fluorescence Y within the range of red fluorescence X X3 to X XS as cells are being measured immediately after staining.

As the green fluorescence increases the average neutrophil green fluorescence is calculated to generate a specific time to produce data for the cell differentiation program. Thus when Y =Ys for X3 X X5 the program switches to the data reduction mode.

The computer builds in memory a two dimensional histogram, i.e., number of cells for each of the two values of fluorescence possible. The first parameter of this histogram is madepropottional to green fluorescence.

The second parameter is made proportional to either red fluorescence or the ratio of red to green fluorescence. The two parameter histogram is partitioned as shown in Table l, below. The values of Xi and Yi are iz'tcd by the operator. The program counts the number of cells in each area art: prints out these counts.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE I A staining composition suitable for use in accordance with the present invention is produced with the following composition: an aqueous solution containing 4 X grams of AO per liter, being adjusted to an osmolarity of approximately one-half of the normal physiologic (isotonic) level through the addition of 4.25 grams of sodium chloride per liter of solution and buffcred at a pH value ofapproximately 7.4. Such buffering can be obtained with phosphates of sodium at a phosphate molarity level of 0.00125 by incorporating 45 milligrams of NaH,PO .H O and 250 milligrams of Na,HPO .7H,O into each liter of solution.

This composition can be produced by the following steps. A l-liter container is partially filled with distilled water and 4.25 grams of sodium chloride may be added together with 0.045 grams of NaH PO.H,O and 0.250 grams of Na HPofll-i O. The mixture is stirred or agitated until the salts go into solution, and the container is then topped up to a total volume of 1 liter by adding additional distilled water. In a separate container, 400 milligrams of acridine orange powder are combined with sufficient distilled water to produce 100 cubic centimeters of acridine orange solution. This combination is stirred or agitated until the acridine orange is dissolved in the water. This results in a clear solution with a reddish-orange tinge of color. One cubic centimeter of 100 cubic centimeters of acridine orange solution is then added to the previously mixed one liter of saline-buffer solution to produce the acridine orange composition described above. The combination with a saline buffer appears to cause some of the acridine orange to form a colloidal suspension.

Preferably, the combined composition is checked with a pH meter, and if further adjustment is necessary, a drop or two of one-tenth normal hydrochloric acid is added to lower the pH, or a drop or two of one-tenth normal sodium hydroxide is added to raise the pH.

This dye composition was used in accordance with the teachings of this invention and was found to protime a very satisfactory result.

EXAMPLE ll Example I is repeated, substituting for the phosphate buffer, a buffer commonly referred to as HEPES (i.e., N-Z-hyd roxy-ethylpiperazine-N -2-ethanesulfonic acid) at a molarity level of 0.005.

EXAMPLE III Example I is again repeated, substituting for the phosphate buffer a buffer commonly referred to as TRIS (i.e., 2-amino-2-(hydroxy-methyl)-l,3- propanediol) at a molarity level of 0.15.

EXAMPLE IV Example I is again repeated with the exception that the amount of acridine orange was quadrupled to provide a concentration of 1.6 X 10" grams per liter. At this concentration, the results are found to be very good in the method of the present invention.

EXAMPLE V Example I is again repeated, except that the concentration of acridine orange is increased to a level of 4 X 10' grams of acridine orange per liter ofsolution, all of the other conditions being as set forth in Example I. This composition provides results whichare satisfactory, but which are not quite as effective as the lower concentrations of acridine orange described in the preceding examples. Accordingly, this is believed to represent the approximate upper limit of acridine orange concentration which is effective in accordance with the t present invention. The staining effect appears to b etoo great to provide good differentiation between different classes of white cells.

EXAMPLE VI Example I is again repeated, except that the acridine orange concentration is reduced to 3 X 10 grams of acridine orange per liter of solution, all other conditions remaining the same as in Example I.

This composition provides results which are satisfactory for the purposes of this invention. Thus, it provides a clear distinction between white cells and other blood particles. However, the results in terms of distinguishing one white cell group from another appear to be somewhat less satisfactory than Example I. Accordingly, this concentration of acridine orange is believed to represent the lower limit of the preferred range of concentration.

EXAMPLE VII Example I is repeated, except the acridine orange concentration is reduced to 4 X l0 grams of acridine orange per liter of solution, all other conditions remaining the same as in Example I.

This composition provides results which are satisfactory for some of the purposes of this invention. Thus, it is satisfactory for providing a clear distinction between white cells and other blood particles. However, the results in terms of distinguishing one white cell group from another are seriously impaired. Thus, it appears that this is the minimum concentration of acridine orange which is truly useful in the practice of the present invention.

In all of the Examples II through VII, the acridine orange may, at least to some extent form a colloid when added to the saline-buffer solution, just as described in connection with Example I.

EXAMPLE VIII Example I is repeated, except that the staining composition is produced so as to have an osmolarity which is approximately 0.3 isotonic through the addition of 2.55 grams of sodium chloride per liter of solution. All other conditions remain the same as in Example l.

This composition provides results which are unsatisfactory for purposes of the present invention due to the fact that some of the white cells are killed before they can be analyzed. Therefore, an osmolarity level which is 0.3 isotonic represents what is believed to be the lower limit of hypotonality which is effective in the present invention.

EXAMPLE IX Example I is repeated, except that the staining composition is produced so as to have an osmolarity which is approximately 0.7 isotonic through the addition of 5.95 grams of sodium chloride per liter of solution. All other conditions remain the same as in Example I.

This composition provides results which are unsatisfactory for purposes of the present invention, due to the fact that the clusters of bright spot fluoroscence signal points are insufficiently separated. Therefore, an osmolarity level which is 0.7 isotonic represents what is believed to be the upper limit of hypotonality which is effective in the present invention.

EXAMPLE X In practicing the method of the present invention, a quantity of 0.10 milliliter of a fresh blood sample is added to l milliliter of the acridine orange diluent staining composition of Example I. The resulting suspension is mildly agitated from time to time and al lowed to stand for a period varying between seconds and several minutes to permit the dye to be taken up by the white cells of the blood sample. The suspension is then introduced into an apparatus having a flow system which provides for a flow of the sample liquid in an exceedingly fine stream through an optical chamber. The stream is so narrow as to be capable of confining the flow so that the individual cells (both red cells and white cells) generally traverse the stream one at a time in single file. In the optical chamber, the cells are caused to pass through a uniform light field provided from a blue laser beam, preferably an argon ion laser at a wavelength of 4880 angstrom units (488 millimicrons). The uniform light field from the laser preferably has a very short dimension in the direction of travel of the cell sample stream. That dimension is of the same order of magnitude as the maximum dimension of an individual cell so that the cells are subjected to radiation one at a time.

Green fluorescent radiation having a range of wavelengths centered at about 5300 angstrom units resulting from the optical excitation of the white cells by the argon laser beam is then detected, and the total number of white cells is counted in terms of optical green fluorescent pulses. This counting is continued for a carefully measured volume of the suspension sample to obtain an exact white cell count per unit of volume.

It has been discovered that at the acridine orange concentrations noted above there is essentially no takeup of acridine orange dye by red cells which therefore remain substantially invisible in the detection method just described. (However, there is an exception to this statement, as noted below). By contrast, each white cell takes up a concentration of the acridine orange dye which results in green fluorescent emission. Thus, a rapid and accurate count of white cells is produced. lrnmature red cells, referred to as reticulocytes, do take up some of the acridine orange dye, but the dye is taken up in such a way that there is substantially no green fluorescene emission from the reticulocytes. Thus, the green fluorescence emission is an accurate basis for distinguishing white cells from all other blood particles.

EXAMPLE XI The method as set forth in Example X is repeated, except that, in addition to detecting the green fluorescence characteristic, the red fluorescence of each cell is detected, at a range of wavelengths in the order of 6500 angstrom units. The red and green signals are optically separated by a dichroic mirror and suitable filters, and amplified by separate photomultiplier tubes. The green signals are used to obtain a total white cell count, while the green and red signals are combined to provide a display pattern upon a cathode ray tube. Thus, the green signal may be used as the vertical dis placement coordinate and the red signal as the horizontal displacement coordinate for a single display spot on the cathode ray tube for each white cell. Various amplitudes of red and green fluorescences from the different white cells are thus displayed upon the cathode ray tube. It is believed that the different green and red fluorescence amplitudes are due to the variation in characteristics of different types of white cells, the white cells having the largest number of granules in the cytoplasm generally displaying the largest red fluorescence signal. However, the chemical composition of various granules among the granulocytes are characteristically different and result in differences in red fluorescence intensity and although the white cells all contain the same amount of DNA, this green fluorescene intensity of the cell nucleus may be modulated in characteristic manners by the differences of avariciousness for the dye in the cell by the granules of various cells resulting in different green fluorescent signals. It has been discovered that each blood sample produces a display pattern having distinct clusters of points indicating the distribution of different types of white cells. Thus, a twodimensional display pattern is produced upon the face of the cathode ray tube which provides qualitative information about the distribution of white cells within the sample. By comparing the patterns produced by blood samples from normal inidviduals with patterns produced by blood samples from individuals who have diseases or infections which cause abnormalities in the balance of white cells, it is quite practical to detect the differences and to quickly recognize, in a qualitative sense, white cell imbalance conditions which are characteristic of particular diseases or infections.

The pattern displayed in connection with the last described example of the method is preferably photographed to provide a permanent record which can be analyzed and studied, and which can be compared with later tests from the same patient.

EXAMPLE Xll Example X1 is repeated, and threshold circuits are employed to select green and red fluorescence signals within individual narrow fields of clusters, and those signals are individually counted for a pre-selected total white cell count sample. The method is then repeated for other settings of the threshold circuits to successively count separate cluster portions of the display corresponding to different classes of white cells. By this means, individual counts of the quantities of white cells of each different type are obtained. Thus, the ratio of the population of each type of white cell to the total white cell count may be obtained. These ratios are preferably presented as percentages.

Multiple threshold circuits may be employed to count several narrow fields of clusters at once.

Since the apparatus operates very rapidly, white cells counting rates as high as L000 cells per second are achievable. previously unattainable accuracies can be realized by counting thousands of white cells of each type from each sample, as contrasted with the usual manual microscope method of counting a very small total quantity in the order of 200 individual cells. Furthermore, since the cells are actually living at the time they are counted, and since they have been undamaged by the use of any procedure to destroy the red cells, the measured cell population corresponds very accurately 1 the cell population in the living blood stream of the patient.

It has been discovered that the staining composition and the methods of the present invention are extremely effective in detecting and counting white cells, in dis tinguishing white cells from red cells on the basis of green fluorescence (essentially ignoring the presence of the red cells), and most particularly in distinguishing the different types of white cells. The different types are distinguishable in the cathode ray tube display by reason of creation of a discrete cluster of points for each distinct type. It has been determined thus far that six classes of white cells, namely, the lymphocytes, monocytes, neutrophils, basophils, eosinophils and immature forms of granulocytes are distinguishable on this basis.

EXAMPLE XlII Example Xl is repeated using a helium-cadmium laser instead of an argon laser. The results are satisfac tory, although the distinctness of some of the oscilloscope clusters are not as well maintained as with the argon ion laser, probably due to the quality of the helium-cadmium laser tested.

EXAMPLE XIV All of the emphasis thus far has been upon the objective of distinguishing white cells from all other particles in the blood, and upon deriving useful information about the white cells. However, the discoveries of the present invention are also useful for deriving other important information about the blood. For instance, the present invention can be employed to detect and count the number of reticulocytes per unit of volume of blood. Retieulocytes are red blood cells which contain a network of granules or filaments representing an immature stage in development. Retieulocytes normally comprise about one percent of the total red blood cells, but this percentage of reticulocytes can change dramatically under abnormal conditions, and such a change may be symptomatic of disease.

It has been observed that the reticulocytes take up acridine orange dye, under the conditions generally outlined in the above examples, to a much greater extent than the other red cells. The uptake of the acridine orange by the other red cells is insignificant, but the re ticulocytes take up enough dye to provide a red fluorescence signal which is of substantially the same magnitude as the lymphocytes class of white cells. However, the reticulocytes do not provide a significant green fluorescence signal. Thus, it is possible to distinguish reticuloeytes from all other blood particles by staining a blood sample with the acridine orange composition of the present invention, and then distinguishing the reticulocytes by excluding from detection all of the green fluorescing white cells, and detecting those remaining cells which have a significant red fluorescence. The following example illustrates this modification of the process.

The process of Example X is repeated, except that the dye concentration is raised to that of Example V and the optical green fluorescent radiation from the white cells is employed as a discrimination signal to exclude any count of the white cells, and with the further exception that in addition to detecting the green fluorescence characteristics, the red fluorescence of each cell is detected at a range of wavelengths in the order of 6,500 angstrom units to thereby select all of those cells having a significant red fluorescence signal, and which at the same time do not produce any green fluo rescence signal. As described in connection with Exam ple XI, the green signal may be used as the vertical dis placement coordinate, and the red signal as the hori zontal displacement coordinate as a basis for setting a low green fluorescence threshold which excludes all of the white cells, and for setting red fluorescence thresholds which particularly select the reticulocytes.

EXAMPLE XV The process of Example XI is repeated using an apparatus of the type schematically represented in FIG. I. The red and green photomultiplier signals after amplification are sampled and held to produce two pulses whose amplitudes are proportional to the magnitudes of the two fluorescent amplitudes. A threshold circuit is connected to the green photomultiplier tube signal and produces a control signal pulse when the green fluorescence is above a fixed value indicating a leukocyte, and activates the sample-hold amplifiers and timing signals for the converters and computer. The two held fluorescence pulses are each converted to a digital representation of the fluorescence values by two analogdigital converters producing two eight-bit numbers in two digital registers. The registers are connected in turn to the input data buss of a computer, such as a Data General. The computer processes the signals as well as input from the operator via a teletype and produces an output on the teletype indicating the differential count,

The computer first monitors the values of green fluorescence Y within the range of red fluorescence X X;, to X X as cells are being measured immediately after staining. As the green fluorescence increases the average neutrophil green fluorescence is calculated to generate a specific time to produce data for the cell differentiation program. Thus when Y Ys for X 4 X 2 X the program switches to the data reduction mode.

The computer builds in memory a two dimensional histogram, i.e., number of cells for each of the two values of fluorescence possible. In our system this grid has a dimensionality 01134 X 64. The first parameter of this histogram is made proportional to green fluorescence. The second parameter is made proportional to either red fluorescence or the ratio of red to green fluorescence.

In a simple differentiation program. 10,000 cells are ead in after Y Yr. to form the histogram of green versus red fluorescence or green versus the ratio of red to green fluorescence. The two parameter histogram is partitioned as shown in Table l, above. The values of X, and Y, are fixed by the operator. The program counts the number of cells in each area and prints out these counts. Also, the first three statistical moments of the data in each area are computed for both parameters to derive indices which can be correlated with specific diseases.

In a more complex program. the lines separating populations are not fixed but are established by the program. To find the proper value for X for example, the data between Y and Y are compressed to generate a one dimensional histogram of the values of red or ratio fluorescence within the green fluorescence range Y to Y There will be a value of fluorescence between the lymphocyte and monocyte populations for which there is a minimum number of cells. This minimum can be determined in several ways, for example, by regression analysis with a parabolic function.

The foregoing examples have been presented for the purpose of illustrating (but not limiting) the compositions, apparatus and methods of the present invention. It will be understood that changes and variations therein may be made without departing from the spirit and scope of the invention as defined in the following claims We claim:

1. A method for distinguishing specific types of cells among white blood cells and other particles within a blood sample, said method comprising the steps of:

a. forming an aqueous diluent solution ofa metachromatic fluorochrome dye, said solution being of hypotonic osmolarity and of physiologically normal pH, and said dye being capable of staining the nuclear material of the white cells to provide fluorescence from the stained nuclear material which is different from the fluorescence of any other blood particle; said hypotonic diluent solution being further characterized in that it can alter the normal rates of dye uptake by the various components of the white cells;

b. staining the vital white cells of a blood sample by suspending the blood sample in the diluent dye solution prepared in step (a);

c. subjecting the suspension formed in step (b) before dye uptake equilibrium is reached therein to illumination from a light source having a radiation wavelength absorbable by the stained cellular material; and

d. distinguishing the various white cells by the magnitudes of their fluorescence emissions.

2. A method for distinguishing white blood cells from other particles within a blood sample and for differentiating the white blood cells as between ncutrophils, eosinophils, basophils, lymphocytes, monocytes, and immature granulocytes, said method comprising the steps of:

a. combining a blood sample with an aqueous solution of acridine orange having a physiologically normal pH factor to form a suspension, said solu' tion having a concentration of acridine orange sufficient to provide a concentration of acridine orange in the suspension within the range of be- '1 "mr' l X and about 4 X 10 grams per liter of suspension, and said solution having a salt concentration corresponding to less than the physiologically normal osmolarity;

b. subjecting the suspension obtained in step (a) to radiation and distinguishing the white cells from all other blood particles by the detection of green fluorescence emitted from individual white cells in response to the excitation from the radiation; and

c. simultaneously detecting the magnitudes of red and green fluorescences emitted from individual white cells in response to excitation from the radiation, and differentially classifying the white cells on the basis of such detection.

3. A method according to claim 2 wherein the blood sample is fresh peripheral blood and wherein said salt concentration within the solution of acridine orange is less than about 7.5 grams per liter of solution.

4. A method according to claim 2 wherein the suspension obtained in step (a) is directed in step (b) in a fine stream through the flow channel of an optical chamber wherein the suspension is subjected to radiation, the fine stream within the flow channel being so narrow as to be capable of confining the flow to substantially a single cell width such that the cells traverse the radiation beam one at a time in single file.

5. A method according to claim 4 wherein the radiation employed in step (b) is a blue laser of wavelength in the neighborhood of 488 millimicrons and is directed toward the suspension stream in the form of a narrow beam substantially transverse to the suspension stream and having a beam dimension in the direction of the suspension stream which is on the same order of magnitude as the maximum dimension of an individual cell.

6. A method according to claim 5 wherein the step of subjecting said suspension to radiation from a blue laser is carried out by energizing an argon ion laser and directing the resulting radiation to said suspension.

7. A method according to claim 4 wherein the step of subjecting said suspension to radiation is carried out by energizing a helium-cadmium laser and directing the resulting radiation to said suspension.

8. A method according to claim 2 wherein the red fluorescence radiation is discriminated by an optical filter which passes wavelengths on the order of 6,500 angstrom units and the green fluorescence radiation is discriminated by an optical filter which passes wavelengths on the order of 5,300 angstrom units.

9. A method according to claim 2 wherein the differences in the magnitudes of red fluorescence and the differences in the magnitudes of green fluorescence emitted from individual cells are detected by convert ing the optical signals into electrical signals and displaying the resultant electrical signals upon the face of a cathode ray tube.

10. A method for making a differential blood analysis of white cells, said method comprising the steps of:

a. combining undiluted and untreated fresh blood with an aqueous solution of acridine orange in the ratio of up to about 1 part of blood to 5 parts of solution to form a suspension, said solution having a concentration in the neighborhood of 4 X 10 grams of acridine orange per liter of solution. said solution containing a buffering agent to maintain the pH factor of the suspension within the normal range for human blood, and said solution having an osmolarity below the normal physiological range for human blood plasma;

b. passing the suspension formed in step (a) through a photoanalysis apparatus and subjecting each white cell in the suspension to radiation;

c. discriminating the white cells from all blood particles contained in the suspension by detecting fluorescent radiation emissions above a predetermined threshold value from each cell in the green portion of the visible spectrum;

d. detecting the magnitudes of the fluorescent radiation emissions from each white cell in the red and green portions of the visible spectrum; and

e. classifying cells based upon the differences in magnitudes of red and green fluorescences measured from the cells.

11. A method according to claim wherein the osmolarity of the acridine orange solution is between 0.3 and 0.7 isotonic.

12. A method according to claim 11 wherein the green radiation signals are employed to derive a total count of the white cells from a measured volume of the suspension.

13. A method according to claim 1] wherein:

the magnitudes of the fluorescent radiation emissions from each cell in the red portion of the visible spectrum are detected by converting the red optical signals to electrical signals and displaying said electrical signals upon the face of a cathode ray tube, the magnitude of the red signals being indicated in terms of the cathode ray tube deflection on one axis; and the magnitudes of the fluorescent radiation emissions from each cell in the green portion of the visible spectrum are detected by converting the green optical signals to electrical signals and displaying said electrical signals upon the face of a cathode ray tube, the magnitude of the green signals being indicated in terms of the cathode ray tube deflection the other axis.

14. A method according to claim 13 wherein a camera is placed over the face of the cathode ray tube to record the pattern of spots produced by said method.

15. A method according to claim 10 wherein:

voltage thresholds are applied to select red fluorescence signals within a narrow field and green fluorescence signals within a narrow field; and

the signals from the overlapping portions of the narrow red and green fields are individually counted for a carefully measured blood sample volume to obtain a quantitative measure of those white cells having the particular characteristics exemplified by overlapping portions of said red and green fields.

16. A method according to claim 13 wherein:

the green fluorescence signals above a predetermined threshold are counted up to a predetermined count value to thereby determine the pas sage of the number of white cells corresponding to that count value;

voltage thresholds are applied to select red fluorescence signals within a narrow field and green fluorescence signals within a narrow field; and

the signals from the overlapping portions of the narrow red and green fields are individually counted during the period of the green fluorescence count to thereby obtain a count ratio of those white cells having the particular characteristics examplified by the field common to the selected narrow red and green fields.

17. A method according to claim 16 wherein the method is repeated with different portions of the same blood sample and with different settings of the voltage thresholds to thereby obtain a series of measurements of cells having different characteristics.

18. A method according to claim 16 wherein the voltage thresholds are applied by parallel threshold circuits to select green fluorescence signals within a selected narrow field and red fluorescence signals within a selected narrow field simultaneously for each of the lymphocyte, monocyte, neutrophil, basophil, eosinophil and immature granulocyte classes of white cells.

19. A method according to claim 18 wherein:

for each of the said classes of white cells and the green fluorescence signals within a selected narrow field and the red fluorescence signals within a selected narrow field are respectively converted to a digital representation of the fluorescence values on a digital register by an analog-digital converter each of said registers being connected to the input data buss of a computer;

the computer monitors, for each of the said classes of white cells, the green fluorescence values corresponding to the red fluorescence signals within said selected narrow field; and

the computer produces a count of the number of cells having fluorescence signals within each selected narrow field of red and green fluorescence signals.

20. A method for making a count of total white cells in a blood sample comprising the steps of:

a. combining a fresh blood sample with an aqueous solution of acridine orange to form a suspension, said solution having a physiologically normal pH factor and a concentration of acridine orange in said suspension in the neighborhood of l0 grams per liter of suspension, and said solution having an osmolarity below the physiologically normal level of human blood;

b. subjecting the suspension obtained from step (a) to radiation from a blue laser and counting the cells from a measured volume of suspension based upon the detection of green fluorescence emitted from each individual cell in response to excitation from the blue laser radiation.

21. A method according to claim 20 wherein the detection of green fluorescence emitted from the cells is discriminated by an optical filter which passes radiation in the neighborhood of 5,300 angstrom units wavelength.

22. A method for classifying vital white blood cells amoung the groups of lymphocytes, monocytes, neutrophils, basophils, eosinophils and immature forms of granulocytes, said method comprising the steps of:

a. combining a fresh blood sample with a hypotonic colloidal solution of a metachromatic fluorochrome dye at a physiologically normal pH; and

b. distinguishing the groups of white blood cells by measuring the magnitudes of metachromatic fluorescences from the individual cells in response to optical radiation.

23. A method for distinguishing specific types of cells among white blood cells and other particles within a blood sample, said method comprising the steps of:

a. forming an aqueous diluent solution ofa metachromatic fluorochrome dye, said solution being capable of staining the nuclear material of the white cells to provide fluorescence from the stained nuc. subjecting the suspension formed in step (b) before dye uptake equilibrium is reached therein to illumination from a light source having a radiation wavelength absorbable by the stained cellular material; and

d. distinguishing the various white cells by the magnitudes of their fluorescence emissions. 

1. A method for distinguishing specific types of cells among white blood cells and other particles within a blood sample, said method comprising the steps of: a. forming an aqueous diluent solution of a metachromatic fluorochrome dye, said solution being of hypotonic osmolarity and of physiologically normal pH, and said dye being capable of staining the nuclear material of the white cells to provide fluorescence from the stained nuclear material which is different from the fluorescence of any other blood particle; said hypotonic diluent solution being further characterized in that it can alter the normal rates of dye uptake by the various components of the white cells; b. staining the vital white cells of a blood sample by suspending the blood sample in the diluent dye solution prepared in step (a); c. subjecting the suspension formed in step (b) before dye uptake equilibrium is reached therein to illumination from a light source having a radiation wavelength absorbable by the stained cellular material; and d. distinguishing the various white cells by the magnitudes of their fluorescence emissions.
 2. A method for distinguishing white blood cells from other particles within a blood sample and for differentiating the white blood cells as between neutrophils, eosinophils, basophils, lymphocytes, monocytes, and immature granulocytes, said method comprising the steps of: a. combining a blood sample with an aqueous solution of acridine orange having a physiologically normal pH factor to form a suspension, said solution having a concentration of acridine orange sufficient to provide a concentration of acridine orange in the suspension within the range of between about 4 X 10 4 and about 4 X 10 2 grams per liter of suspension, and said solution having a salt concentration corresponding to less than the physiologically normal osmolarity; b. subjecting the suspension obtained in step (a) to radiation and distinguishing the white cells from all other blood particles by the detection of green fluorescence emitted from individual white cells in response to the excitation from the radiation; and c. simultaneously detecting the magnitudes of red and green fluorescences emitted from individual white cells in response to excitation from the radiation, and differentially classifying the white cells on the basis of such detection.
 3. A method according to claim 2 wherein the blood sample is fresh peripheral blood and wherein said salt concentration within the solution of acridine orange is less than about 7.5 grams per liter of solution.
 4. A mEthod according to claim 2 wherein the suspension obtained in step (a) is directed in step (b) in a fine stream through the flow channel of an optical chamber wherein the suspension is subjected to radiation, the fine stream within the flow channel being so narrow as to be capable of confining the flow to substantially a single cell width such that the cells traverse the radiation beam one at a time in single file.
 5. A method according to claim 4 wherein the radiation employed in step (b) is a blue laser of wavelength in the neighborhood of 488 millimicrons and is directed toward the suspension stream in the form of a narrow beam substantially transverse to the suspension stream and having a beam dimension in the direction of the suspension stream which is on the same order of magnitude as the maximum dimension of an individual cell.
 6. A method according to claim 5 wherein the step of subjecting said suspension to radiation from a blue laser is carried out by energizing an argon ion laser and directing the resulting radiation to said suspension.
 7. A method according to claim 4 wherein the step of subjecting said suspension to radiation is carried out by energizing a helium-cadmium laser and directing the resulting radiation to said suspension.
 8. A method according to claim 2 wherein the red fluorescence radiation is discriminated by an optical filter which passes wavelengths on the order of 6,500 angstrom units and the green fluorescence radiation is discriminated by an optical filter which passes wavelengths on the order of 5,300 angstrom units.
 9. A method according to claim 2 wherein the differences in the magnitudes of red fluorescence and the differences in the magnitudes of green fluorescence emitted from individual cells are detected by converting the optical signals into electrical signals and displaying the resultant electrical signals upon the face of a cathode ray tube.
 10. A method for making a differential blood analysis of white cells, said method comprising the steps of: a. combining undiluted and untreated fresh blood with an aqueous solution of acridine orange in the ratio of up to about 1 part of blood to 5 parts of solution to form a suspension, said solution having a concentration in the neighborhood of 4 X 10 3 grams of acridine orange per liter of solution, said solution containing a buffering agent to maintain the pH factor of the suspension within the normal range for human blood, and said solution having an osmolarity below the normal physiological range for human blood plasma; b. passing the suspension formed in step (a) through a photoanalysis apparatus and subjecting each white cell in the suspension to radiation; c. discriminating the white cells from all blood particles contained in the suspension by detecting fluorescent radiation emissions above a predetermined threshold value from each cell in the green portion of the visible spectrum; d. detecting the magnitudes of the fluorescent radiation emissions from each white cell in the red and green portions of the visible spectrum; and e. classifying cells based upon the differences in magnitudes of red and green fluorescences measured from the cells.
 11. A method according to claim 10 wherein the osmolarity of the acridine orange solution is between 0.3 and 0.7 isotonic.
 12. A method according to claim 11 wherein the green radiation signals are employed to derive a total count of the white cells from a measured volume of the suspension.
 13. A method according to claim 11 wherein: the magnitudes of the fluorescent radiation emissions from each cell in the red portion of the visible spectrum are detected by converting the red optical signals to electrical signals and displaying said electrical signals upon the face of a cathode ray tube, the magnitude of the red signals being indicated in terms of the cathode ray tube deflection on one axis; and the magnitudes of the fluOrescent radiation emissions from each cell in the green portion of the visible spectrum are detected by converting the green optical signals to electrical signals and displaying said electrical signals upon the face of a cathode ray tube, the magnitude of the green signals being indicated in terms of the cathode ray tube deflection the other axis.
 14. A method according to claim 13 wherein a camera is placed over the face of the cathode ray tube to record the pattern of spots produced by said method.
 15. A method according to claim 10 wherein: voltage thresholds are applied to select red fluorescence signals within a narrow field and green fluorescence signals within a narrow field; and the signals from the overlapping portions of the narrow red and green fields are individually counted for a carefully measured blood sample volume to obtain a quantitative measure of those white cells having the particular characteristics exemplified by overlapping portions of said red and green fields.
 16. A method according to claim 13 wherein: the green fluorescence signals above a predetermined threshold are counted up to a predetermined count value to thereby determine the passage of the number of white cells corresponding to that count value; voltage thresholds are applied to select red fluorescence signals within a narrow field and green fluorescence signals within a narrow field; and the signals from the overlapping portions of the narrow red and green fields are individually counted during the period of the green fluorescence count to thereby obtain a count ratio of those white cells having the particular characteristics examplified by the field common to the selected narrow red and green fields.
 17. A method according to claim 16 wherein the method is repeated with different portions of the same blood sample and with different settings of the voltage thresholds to thereby obtain a series of measurements of cells having different characteristics.
 18. A method according to claim 16 wherein the voltage thresholds are applied by parallel threshold circuits to select green fluorescence signals within a selected narrow field and red fluorescence signals within a selected narrow field simultaneously for each of the lymphocyte, monocyte, neutrophil, basophil, eosinophil and immature granulocyte classes of white cells.
 19. A method according to claim 18 wherein: for each of the said classes of white cells and the green fluorescence signals within a selected narrow field and the red fluorescence signals within a selected narrow field are respectively converted to a digital representation of the fluorescence values on a digital register by an analog-digital converter, each of said registers being connected to the input data buss of a computer; the computer monitors, for each of the said classes of white cells, the green fluorescence values corresponding to the red fluorescence signals within said selected narrow field; and the computer produces a count of the number of cells having fluorescence signals within each selected narrow field of red and green fluorescence signals.
 20. A method for making a count of total white cells in a blood sample comprising the steps of: a. combining a fresh blood sample with an aqueous solution of acridine orange to form a suspension, said solution having a physiologically normal pH factor and a concentration of acridine orange in said suspension in the neighborhood of 10 3 grams per liter of suspension, and said solution having an osmolarity below the physiologically normal level of human blood; b. subjecting the suspension obtained from step (a) to radiation from a blue laser and counting the cells from a measured volume of suspension based upon the detection of green fluorescence emitted from each individual cell in response to excitation from the blue laser radiation.
 21. A method according to claim 20 wherein the detection of green fluorescence emitted from the cells Is discriminated by an optical filter which passes radiation in the neighborhood of 5,300 angstrom units wavelength.
 22. A METHOD FOR CLASSIFYING VITAL WHITE BLOOD CELLS AMOUNG THE GROUP OF LYMPHOCYTES, MONOCYTES, NEUTROPHILIS, BASOPHILS, EOSINOPHILS AND IMMATURE FORMS OF GRANULOCYTES, SAID METHOD COMPRISING THE STEP OF: A. COMBINING A FRESH BLOOD SAMPLE WITH A HYPOTONIC COLLOIDAL SOLUTION OF A METACHROMATIC FLUOROCHROME DYE AT A PHYSIOLOGICALLY NORMAL PH; AND B. DISTINGUISHING THE GROUPS OF WHITE BLOOD CELLS BY MEASURING THE MAGNITUDES OF METACHROMATIC FLUORESCENCES FROM THE INDIVIDUAL CELLS IN RESPONSE TO OPTICAL RADIATION.
 23. A method for distinguishing specific types of cells among white blood cells and other particles within a blood sample, said method comprising the steps of: a. forming an aqueous diluent solution of a metachromatic fluorochrome dye, said solution being capable of staining the nuclear material of the white cells to provide fluorescence from the stained nuclear material which is different from the fluorescence of any other blood particle; said solution being further characterized in that it is capable of altering the differential rates of dye uptake by the various components of the white cells; b. staining the vital white cells of a blood sample by suspending the blood sample in the diluent dye solution prepared in step (a); c. subjecting the suspension formed in step (b) before dye uptake equilibrium is reached therein to illumination from a light source having a radiation wavelength absorbable by the stained cellular material; and d. distinguishing the various white cells by the magnitudes of their fluorescence emissions. 